ABSTRACT Rheumatoid arthritis (RA) is a common and chronic autoimmune arthritis that causes erosive joint damage and deformities, with increased risk for disability and reduced life expectancy. Recent new therapies have significantly improved disease control but remission remains uncommon and new and better treatments are needed. We have recently discovered a previously unrecognized role for magnesium in the regulation of arthritis. Specifically, a short-term experimental low magnesium diet was highly protective reducing arthritis severity and histologic joint damage in pristane-induced arthritis (PIA) in rats and collagen-induced arthritis (CIA) in mice, two well-established models or RA. Our observations suggest that the arthritis-protective effect involves a magnesium-dependent interference with Treg, Tr1 or Th17 cell development and/or localization to the joint. The specific aims are: AIM 1. To determine the role of magnesium on Treg, Tr1 and Th17 T cell differentiation and in fibroblast-like synoviocytes (FLS). Subaim 1. To characterize the effect of reduced magnesium conditions on in vitro Treg, Tr1 and Th17 T cell differentiation and function. Subaim 2. To determine the role of specific magnesium channels in Treg, Tr1 and Th17 differentiation. The role of specific magnesium channels on in vitro Treg, Tr1 and Th17 differentiation will be examined in experiments with siRNA knock-down, and with antagonists and agonists. Subaim 3. The role of magnesium in fibroblast-like synoviocyte (FLS) production of of pro-inflammatory mediators. We will examine whether low concentrations of magnesium reduce the RA FLS production of inflammatory factors including those required for the influx of Th17 and other cells into the synovial tissues, and whether a specific magnesium channel accounts for that. AIM 2. In vivo effect of a short-term experimental low magnesium diet on established arthritis, on the development of protective and pathogenic T cell subsets, and on the intestinal microbiome. Subaim 1. To determine the effect of a short-term low magnesium diet initiated after the onset of disease on arthritis severity and joint damage. It is hypothesized that a low-magnesium diet initiated after the onset of arthritis (CIA) will significantly reduce disease severity and histologic joint damage. The role of specific magnesium channels will be examined with inhibitors. Subaim 2. To determine the role of reduced magnesium conditions on Treg, Tr1 and Th17 cell differentiation in vivo. Will examine the effect of low and high magnesium diets on the numbers of Treg, Tr1 and Th17 cells in arthritis induced in mice with inducible IL17-eGFP/Foxp3-mRFP. Submaim 3. Effect of reduced magnesium conditions on the gut microbiome. We have hypothesized that the low magnesium diet protective effect could be related to changes induced by the diet in the gut microbiome and will comprehensively examine that.